Proteasomes are central to the quality control and regulated turnover of proteins and, thus, are major players in a variety of diseases which impact human health. The haloarchaeon Haloferax volcanii provides an excellent model system for understanding how these multisubunit complexes function as subtypes in the recognition and degradation of proteins in the absence of ubiquitin. This application is based on the identification of proteasome substrates by fluorescent reporter protein analysis and by differential proteomics of proteasome mutant and wild type strains. It is also based on the subunit topology of proteasome subtypes including 20S proteasomes of different alpha subunit composition and hetero- and homo-oligomeric forms of proteasome-activating nucleotidase AAA proteins. Furthermore, it is based on the growth-dependent differential regulation of the mRNA and protein levels of the subunits of these proteasomal subtypes as cells transition from log to stationary phase. This application will test the following hypotheses: (a) Subtypes of AAA ATPases recognize overlapping as well as unique sets of substrates for proteasome-mediated degradation, (b) 20S core particle subtypes of different alpha subunit composition differ in their affinity for these AAA ATPase subtypes, (c) The levels and post-translational modification of the AAA ATPases and alpha proteins are regulated to influence the timing and specificity of protein turnover. Thus, various combinations of AAA ATPase and 20S proteasome subtypes are proposed to form a network for the regulated turnover of proteins in the cell. The proposed combination of genetic and biochemical analysis will identify proteasomal substrates, define how proteasomal subtypes mediate recognition of these substrates, and elucidate the mechanisms involved in regulating the levels and posttranscriptional modification of the components of these proteasome subtypes in H. volcanii.